Microbial load in unpasteurized milk obtained from selected milk outlets in Kawempe division
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MedDocs Publishers
ISSN: 2637-4927
Annals of Biotechnology
Open Access | Research Article
Microbial load in unpasteurized milk obtained
from selected milk outlets in Kawempe division
of Kampala, Uganda
John N Kateregga1; Collins Atuheire1; Esther Nabatta1; Samuel Majalija1; James G Ndukui 1,2*
1
College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University-Kampala, Uganda
2
Department of Medical Laboratory Sciences, College of Health Sciences, Jomo Kenyatta University of Agriculture and Technology,
Nairobi, Kenya.
*Corresponding Author(s): James G Ndukui Abstract
Department of Medical Laboratory Sciences, College Background: Milk consumption and demand has in-
of Health Sciences, Jomo Kenyatta University of Agri- creased drastically over the past years in response to ex-
panding human population in Uganda and world at large.
culture and Technology, Nairobi, Kenya.
Despite this, many residents of Kampala city obtain milk
Email: ndukuiga@gmail.com from shops supplied by milk vendors, who in turn, collect
it directly from farms. The microbial load of this unpasteur-
ized milk is not known, yet it could expose the population to
milk-borne diseases. The purpose of this study was to deter-
mine the level of bacterial contamination of unpasteurized
Received: Nov 06, 2018 milk sold in different milk outlets in Kawempe Division, one
Accepted: Mar 25, 2019 of the five divisions of Kampala City.
Published Online: Mar 29, 2019 Methods: This was a cross-sectional study in which 50
Journal: Annals of Biotechnology milk samples were collected from bulk tanks in different
Publisher: MedDocs Publishers LLC locations of Kawempe Division i.e. Kamwokya, Kalerwe,
Mulago, Bwaise and Kawempe. The samples were taken
Online edition: http://meddocsonline.org/ to the Central Diagnostic Lab of the College of Veterinary
Copyright: © Ndukui JG (2019). This Article is distributed Medicine, Animal Resources and Biosecurity of Makerere
under the terms of Creative Commons Attribution 4.0 University and cultured using suitable media. Total aerobic
International License counts, Staphylococcus aureus counts, coliform as well as
E. coli and Salmonella counts were determined. The preva-
lence of Brucellain the milk samples was determined using
Keywords: Unpasteurized milk; Total plate counts; S aureus; the Brucella milk ring (Rose Bengal) test. The bacteriologi-
coliforms; Brucella cal indices were compared with UNBS and WHO acceptable
standards. ANOVA was used to compare levels of contami-
nation in various locations. All differences were considered
Abbreviations: Cfu/g: Colony Forming Units Per Gram; DDA: significant at p
MedDocs Publishers
Conclusions: This study has shown that the raw milk Materials and methods
sold in outlets of Kawempe division does not meet the set Study design
standards and hence not safe for human consumption. Food
safety authorities should be strengthened and more restric- This was a cross-sectional study in which the level of con-
tions put on the sale of low quality milk. tamination of unpasteurized milk was determined in randomly
collected samples from the different milk collection outlets. Fif-
Background ty samples were collected from the five selected milk vending
outlets in Mulago, Kawempe, Kalerwe, Bwaise, and Kamwokya
Milk and its products are excellent high quality foods provid- parishes of Kawempe Division of Kampala city. Total aerobic
ing both culinary and nutritional values. The constituents may counts, Staphylococcus aureus counts, coliform as well as E.
vary with breed, type of feed, stage of lactation, season and age coli and Salmonella counts were determined. The prevalence
of cow among others, and also individuals of the same breed. of Brucella in the milk samples was determined using theRose
Thus, it serves as an excellent medium for bacterial growth Bengal test.
where under inappropriate conditions it acts as a carrier for
disease causing pathogens from cows to humans (zoonosis). Sample collection and processing
Freshly drawn milk from the udder of a healthy cow should The samples (50 in total) were purchased from the differ-
be free from micro-organisms. However, contaminations have ent milk vending outlets in a systematic simple random method.
been reported and this has been attributed to the movement of The sample size was determined by the standard formula [11]
pathogens up the teat canal and/or their presence at the lower and was based on the assumption that 50% of milk sold by the
ends of the teats [1,3]. Some studies have also showed that milk vendors is unsuitable. Samples were collected in sample bottles
contains low numbers of commensal micro-organisms less than and placed under ice in a cool box and immediately transported
500 bacteria per ml, usually the coagulase negative, nonpatho- to Central Diagnostic Laboratory, College of Veterinary Medi-
genic micrococci and streptococci although coliform bacteria cine, Animal Resources and Biosecurity of Makerere University
are also common [3,4]. for analysis. Samples were collected in such a way that 500ml
Microbial contamination of milk depends on the storage of milk were picked from 10 different vending outlets in each
temperature, the time elapsing before collection and the initial parish. Using a sterile pipette tip, the milk was serially diluted
microflora [5]. When milk is cooled toMedDocs Publishers
and coagulase [14]. (10%), Kamwokya (10%) and lastly Kalerwe (0%) as shown in
Table 1.
Detection of salmonella species
Discussion
The procedure was done basing on ISO 6579: 2003 method-
ologies. Hundred (100) ul of the original sample was inoculated The total aerobic count reported in the study was generally
into 9.9ml of Rappaport media (Oxoid limited, England), incu- high for all the five study areas with Kalerwe having the highest
bated at 42oC for 24 hours then followed by inoculation onto whereas Mulago having the least this could probably be due
XLD agar (Mast Group Limited, UK) with further incubation at to the high numbers of rubbish heaps that originate from the
370C for 24 hours. Red yellow colonies with black centers were market. The study corroborates with [17] which reported an
observed. A colony with the distinct features was then picked exceptionally high total aerobic as well as coliform counts. The
and sub-cultured then confirmed using Indole, methyl red, cit- high total aerobic counts were probably due to poor hygiene
rate and hydrogen sulphide production [13-15]. and management practices whereby the milk was kept at tem-
peratures higher than 4 ̊C, milking of very dirty cows and in ad-
Milk ring test (MRT) for brucellosis dition the cows could have been suffering from mastitis [18].
The test was carried out by adding 30ul of the Brucella anti- Kamwokya had the highest staphylococcus counts whereas Mu-
gen to 2ml of the unpasteurized milk then incubated at 370C for lago had the lowest counts (Table 4) which could probably be
1 hour. A blue ring above a white milk column indicated positive due to the fact the milk received in Kamwokya was got from
whereas no ring indicated a negative test [16]. mastitis infected herds.
Quality management Staphylococcus aureus is often found in raw milk and its
products due to contamination caused by poor hygiene condi-
The systematic random sampling method was used to collect tions or the origin of the milk, which can come from mastitic
a representative sample from the milk sold. Aseptic techniques cows. According to a study done by the National Research Insti-
were employed during processing to prevent any possible for- tute in Poland, 32% the bulk tank milk contained Staphylococ-
eign contamination. Proper labeling and identification was done cus aureus. This prevalence being much lower than that of this
for the different samples going to be processed. The media was study which reported a prevalence of 40%-100% could be due
prepared and sterilized by autoclaving at 121ºC for 15 minutes to variation in environmental conditions or better enforcement
before pouring it on the petri dishes to cool. The samples were of milk hygiene standards in developed countries.
processed as soon as they are collected to prevent any disrup-
tion to its integrity. The very high counts in Bwaise could probably be due to its
location in a swampy area. The high coliform count in the dif-
Data analysis ferent areas could probably also be due to poor hygiene prac-
The results were recorded in Microsoft Excel software and tices whereby the milk gets into contact with feces of healthy
exported to SPSS.16.0 for analysis. In this case ANOVA and chi animals, use of equipment that are not cleaned thoroughly,
square statistical tests were performed. Then tables were drawn milking of dirty cows or cows washed with contaminated wa-
to demonstrate trends and comparisons of the results. The bac- ter. Since coliforms are mastitis causative agents it could also be
teriological indices determined were compared with acceptable due to milking of cows infected with mastitis. Studies in Tanza-
levels set by the UNBS and WHO. nia by Swai (2011) reported a coliform count of 3.0x106 which
is much lower than that reported in this study. This could be
Ethical consideration due to the good hygiene practices and enforcement exercised
in Tanzania. According to Bramley and Mackinnon (1990), coli-
An ethical clearance was sought from College of Veterinary forms multiply rapidly in the environment; therefore their pres-
Medicine, Animal Resources and Biosecurity Ethical Review ence in high numbers in food products is conclusive of improper
Committee. In addition, the study was further approved by handling and contamination. E. coli is the most prominent fecal
student’s research review committee of the College. coliform and its prevalence is indicative of fecal contamination
Results of the milk. E. coli is a reliable parameter for fecal contamina-
tion of water sources and hence its presence in milk is not only
The mean total aerobic counts ranged between 7.3x107cfu/ an indicator for fecal contamination but also an indicator for
ml and 1.67x108cfu/ml. The mean count was highest in samples over all sanitary condition of the dairy farms and the different
got from Kalerwe and lowest in samples got from Mulago (Table selling points (bulk tanks). With revolutionary advances in mod-
1). However there was no significant difference (p= 0.515) in ern medicine, there has been heavy reliance on the availability
the mean counts of samples from all places as shown in Table 4. of effective antibiotics to manage infections and enable invasive
The total aerobic counts of all the milk samples obtained from surgery due to emergence of antibiotic-resistant bacteria, how-
all places did not meet the UNBS standards for milk meant for ever novel approaches are necessary to prevent the formation
human consumption. of biofilms on sensitive surfaces such as antimicrobial nanoma-
The mean Staphylococcus aureus counts ranged from terials that are derived from biological polymers or that rely on
1.97x105 cfu/ml to 1.97x108cfu/ml. Results showed that sam- the incorporation of natural compounds with antimicrobial ac-
ples obtained from Kawempe had the highest mean counts and tivity in nanofibers made from synthetic materials is timely, that
those from Mulago had the lowest mean counts. Comparison of will help to hinder these micro-organisms [19].
mean counts of the samples from the different places showed According to studies elsewhere, the prevalence of E. coli,
a significant difference (p=0.039, ANOVA) in Staphylococcus au- especially the Enterohemorrhagic E.coli (EHEC), strain is usually
reus counts (Table 4). Mulago area recorded the highest num- very low, less than 1%. For example in July 2007, US Department
ber of samples that passed the UNBS standard for Staphylococ- of Agriculture (USDA) reported that nearly a quarter of the raw
cus aureus counts (60%), followed by Kawempe (20%), Bwaise milk collected from 861 farms in 21 states contained bacteria
Annals of Biotechnology 3MedDocs Publishers
linked to illness. Among the results 3% of the samples had possibly leading to more Brucella free herds.
Salmonella and 4% had types of E. coli that cause diarrhea and
gastrointestinal illness. Less than 1% had the most dangerous Conclusion
forms of E. coli. However in the presence of unhygienic
The study shows that there is a considerable level of bacterial
handling practices of milk and the numerous opportunities for
contamination in raw milk especially E. coli. Most of the milk
contamination from the producer to the consumer, the potential
samples did not meet the safety criteria recommended by the
for their propagation and dissemination is real. According to
World Health Organization and the Uganda National Bureau of
Grimaud (2008), Enterohemorrhagic E.coli (EHEC) was present
Standards. These levels may be capable of causing disease to
in raw milk in Kampala city which was in agreement with Grace
humans which is therefore a health risk to milk consumers. This
(2008), who indicated that EHEC exists in Kampala city and
calls for measures by the dairy farmers, milk transporters and
hence Kampala milk is unsafe. However according to [20], the
sellers as well as health inspectors to minimize this problem.
prevalence of EHEC was less than 1%.
Acknowledgements
Kawempe had the highest salmonella counts. Salmonella
contamination of milk could probably be due to poor sanitation We thank the staff of the Central Diagnostic Laboratory -CO-
in terms of equipment which could have got into contact VAB for their cooperation and assistance during laboratory anal-
with fecal matter. Salmonella gets into milk majorly through ysis of the samples, especially Mr.Steven Kakooza. This research
contamination with both human and animal feces; it can also get was financed by internally raised resources by the researchers.
into milk through handling teats with dirty hands by the milkers.
According to studies in Tanzania [21], Salmonella prevalence Authors’ contributions
was reported in 10.1% which is lower than the prevalence of JNK, EN, JGN, and MS conceptualized the project, performed
salmonella reported in this study. This could indicate higher most of the laboratory experiments and wrote the manuscript.
levels of poor hygiene and fecal contamination in Kampala. CA assisted in statistically analysing the data. JGN assisted in
According to [22], 56% of the samples at the collection points drafting and finalizing the manuscript. All authors read and ap-
in Tanzania were positive for Brucella; a prevalence value much proved the final manuscript.
higher than reported in this study. This could probably be due
to high levels of vaccination in Uganda compared to Tanzania
Tables
Table 1: Conformity of microbial counts of milk samples with UNBS standards
Bwaise Kawempe Kalerwe Mulago Kamwokya
Parameter
Pass Fail Pass Fail Pass Fail Pass Fail Pass Fail
Total aerobic counts 0% 100% 0% 100% 0% 100% 0% 100% 0% 100%
S. aureus counts 10% 90% 20% 80% 0% 100% 60% 40% 10% 90%
E. coli counts 0% 100% 0% 100% 0% 100% 0% 100% 0% 100%
Coliform counts 20% 80% 0% 100% 0% 100% 0% 100% 0% 100%
Key: The mean total coliform counts ranged from 1.9690x10 cfu/ml to 4.3120x10 cfu/ml with Kawempe having the lowest mean
8 8
counts and Mulago having the highest value. Comparison of the different parishes showed that there was no significant difference
the mean total coliform counts (p=0.700). The mean E. coli counts ranged from 1.24x108cfu/mlto 3.63x108cfu/ml. Kawempe had
the highest mean counts and Kalerwe had the lowest mean counts. Comparison of mean counts of samples from the different
places showed that there was no significant difference (p=0.466) in the mean of E. coli (Table 2).
Table 2: Mean microbial counts in milk samples from the different parishes
Sampling sites
Parameter(cfu/ml) P-value
Bwaise Kawempe Kalerwe Mulago Kamwokya
Total aerobic counts 1.0320x109 ±7.51x108 1.2740x109±7.85x108 1.6710x109±2.38x109 7.3800x108±3.59x108 1.0670x109±4.80x108 0.515
S. aureus counts 1.9870x106 ±4.59x106 1.9690x108±1.76x108 1.3932x108±3.42x108 1.1080x106±2.75x106 2.2760x108±2.42x108 0.039
E. coli counts 2.9124x108±3.44x108 3.6300x108±5.52x108 1.2350x108±6.96x107 2.3750x108±2.30x108 1.6500x108±1.59x108 0.466
Coliform counts 3.2280x108±3.74x108 1.9690x108±1.76x108 3.1000x108±3.42x108 4.3120x108±6.47x108 2.2760x108±2.42x108 0.700
Key: Out of the 50 analyzed milk samples, 52% contained Salmonella. Samples from Bwaise and Mulago contained the highest percentage of
Salmonella (80%) this was followed by Kawempe which had 10%, Kamwokya and Kalerwe samples did not have any Salmonella. There was a
significant association (p=0.000, Chi Square) between sampling locations and Salmonella prevalence (Table 3).
Annals of Biotechnology 4MedDocs Publishers
Table 3: Salmonella prevalence in milk samples from the different sampling sites
Salmonella spps status
Location Samples (N)
Present Absent
Bwaise 10 80.0% (8) 20% (2)
Kamwokya 10 0% (0) 100.0% (10)
Mulago 10 80.0% (8) 20.0% (2)
Kawempe 10 100.0% (10) 0% (0)
Kalerwe 10 0% (0) 100.0% (10)
TOTAL 52.0% 48.0%
50
Key: X2: 37.179, P: 0.00: spps: Species, N: Sample Size
The samples from all different collection points did not contain any Brucella antibodies as shown in Table 4.
Table 4: Brucella antibody prevalence in milk samples from the different parishes
Brucella Status
Place Total
Present Absent
Bwaise 0% 100.0% 100.0%
Kamwokya 0% 100.0% 100.0%
Mulago 0% 100.0% 100.0%
Kawempe 0% 100.0% 100.0%
Kalerwe 0% 100.0% 100.0%
Total 0% 100.0% 100.0%
Key: %:Percentage
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